Potential Red-Flag Identification of Colorectal Adenomas with Wide-Field Fluorescence Molecular Endoscopy

Elmire Hartmans, Jolien J J Tjalma, Matthijs D Linssen, Pilar Beatriz Garcia Allende, Marjory Koller, Annelies Jorritsma-Smit, Mariana E Silva de Oliveira Nery, Sjoerd G Elias, Arend Karrenbeld, Elisabeth G E de Vries, Jan H Kleibeuker, Gooitzen M van Dam, Dominic J Robinson, Vasilis Ntziachristos, Wouter B Nagengast, Elmire Hartmans, Jolien J J Tjalma, Matthijs D Linssen, Pilar Beatriz Garcia Allende, Marjory Koller, Annelies Jorritsma-Smit, Mariana E Silva de Oliveira Nery, Sjoerd G Elias, Arend Karrenbeld, Elisabeth G E de Vries, Jan H Kleibeuker, Gooitzen M van Dam, Dominic J Robinson, Vasilis Ntziachristos, Wouter B Nagengast

Abstract

Adenoma miss rates in colonoscopy are unacceptably high, especially for sessile serrated adenomas / polyps (SSA/Ps) and in high-risk populations, such as patients with Lynch syndrome. Detection rates may be improved by fluorescence molecular endoscopy (FME), which allows morphological visualization of lesions with high-definition white-light imaging as well as fluorescence-guided identification of lesions with a specific molecular marker. In a clinical proof-of-principal study, we investigated FME for colorectal adenoma detection, using a fluorescently labelled antibody (bevacizumab-800CW) against vascular endothelial growth factor A (VEGFA), which is highly upregulated in colorectal adenomas. Methods: Patients with familial adenomatous polyposis (n = 17), received an intravenous injection with 4.5, 10 or 25 mg of bevacizumab-800CW. Three days later, they received NIR-FME. Results: VEGFA-targeted NIR-FME detected colorectal adenomas at all doses. Best results were achieved in the highest (25 mg) cohort, which even detected small adenomas (<3 mm). Spectroscopy analyses of freshly excised specimen demonstrated the highest adenoma-to-normal ratio of 1.84 for the 25 mg cohort, with a calculated median tracer concentration in adenomas of 6.43 nmol/mL. Ex vivo signal analyses demonstrated NIR fluorescence within the dysplastic areas of the adenomas. Conclusion: These results suggest that NIR-FME is clinically feasible as a real-time, red-flag technique for detection of colorectal adenomas.

Keywords: near-infrared fluorescence.; optical molecular imaging; spectroscopy; vascular endothelial growth factor a.

Conflict of interest statement

Competing Interests: G.M.v.D. and W.B.N. received an unrestricted research grant made available to the institution for the development of optical molecular imaging from SurgVision BV ('t Harde, the Netherlands). G.M.v.D. and V.N. are members of the scientific advisory board of SurgVision BV. Other authors declare no competing financial interests.

Figures

Figure 1
Figure 1
Study design. (A) Intravenous administration of the fluorescent tracer bevacizumab-800CW, three days later followed by VEGFA-targeted fluorescence endoscopy. (B) Ex vivo fluorescent signal analyses: 1) quantification of the fluorescence signals with MDSFR/SFF spectroscopy, performed on fresh resected tissue, and 2) qualitative evaluation of tracer distribution performed on FFPE tissue. CR, colorectal; FFPE, formalin-fixed and paraffin-embedded; H&E, hematoxylin and eosin; IHC, immunohistochemistry; MDSFR/SFF, multi-diameter single fiber reflectance and single fiber fluorescence; NIR-FME, near-infrared fluorescence molecular endoscopy; VEGFA, vascular endothelial growth factor A.
Figure 2
Figure 2
Wide-field VEGFA-targeted fluorescence endoscopy. Three adenomas per tracer-dose cohort. The clinical white-light images gained with a HD video endoscope (first column), combined with representative overlay images (second column) and NIR fluorescence images (third column) gained with the NIR fiber bundle. The overlay images are automatically generated by the software, showing the highest fluorescence intensities in bright green and the very low fluorescence intensities as absent. The fluorescence images were taken with different exposure times.
Figure 3
Figure 3
Real-time in vivo NIR-FME images. Adenomas from the 25 mg dose cohort demonstrating the ability of the NIR-FME system to visualize small and flat adenomas at video frame rate (10 frames per second). The white arrow indicates a second small adenoma. The overlay images are automatically generated by the software, showing the highest fluorescence intensities in bright green and the very low fluorescence intensities as absent.
Figure 4
Figure 4
Fluorescence quantification by MDSFR/SFF spectroscopy. Box plot (median, 10-90 percentile) showing the intrinsic fluorescence (Q.µfa,x) per bevacizumab-800CW tracer-dose cohort for both LGD containing adenomas and the normal colorectal tissue (biopsies). For all dose cohorts, a significant difference in fluorescence intensity can be observed between the benign and premalignant tissue, which increases with increasing tracer dosages. Note that the fluorescence in the normal tissue stays constant in the 10 and 25 mg cohorts, regardless of the tracer dose used. The median adenoma-to-normal ratio of intrinsic fluorescence was 1.84 for the 25 mg cohort. * = P < 0.05; ** = P < 0.001.
Figure 5
Figure 5
Ex vivo fluorescent signal analyses. (A) Representative NIR fluorescence flatbed scan of a fluorescent adenoma (10 mg dose cohort), containing both dysplasia and normal colon crypts in the same section (HE staining). The fluorescence scan and interactive surface plot demonstrate that the fluorescence intensities are the highest at the sites of dysplasia, a phenomenon that was observed in all three tracer-dose cohorts. (B) Three representative fluorescence microscopy images, demonstrating an observable difference in fluorescence intensities between the three dose cohorts: the 4.5 mg cohort shows a lower signal in the 800 nm channel, compared to the two higher dose cohorts. Fluorescence microscopy did not show a clear difference between the two highest dose cohorts (10 mg vs 25 mg). The left column represents the fluorescence of the tracer (800 nm channel), the middle column shows Hoechst staining of the nuclei and third column displays an overlay of the previous two channels. (C) Representative microscopy images of one adenoma of the 25 mg dose cohort, showing a clear difference in NIR fluorescent signal between areas containing dysplasia and areas containing normal colon crypts; the areas can be distinguished based on the appearance of the crypts, since stacking of the nuclei is typical for dysplasia.
Figure 6
Figure 6
VEGFA immunohistochemistry results. (A) Box plot (median, 10-90 percentile) and bar graph, both presenting VEGFA IHC results (H-score) of adenomatous colorectal polyps (LGD and normal crypts) and normal colorectal biopsies; a clear difference in H-score can be observed between the adenomatous crypts and the normal surrounding tissue and normal biopsies. (B) Representative images illustrating the clear difference in VEGFA staining intensities (brown) between dysplastic and normal crypts (areas within dashed yellow lines display normal crypts).

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